We have previously demonstrated that both CD8+ and CD4+ human tumor infiltrating lymphocytes (TIL) can manifest XHC-restricted recognition of tumor associated antigens (Ag). This has been shown for human melanomas as well as colon carcinomas, breast carcinomas, and lymphomas. Current projects focus on the precise characterization of the recognized Ag for developing vaccination strategies. 1.Immunotherapy of melanoma with adoptive transfer of TIL: Correlation of in vitro characteristics of transferred cells with clinical response. A 2-year study of 41 Stage IV melanoma patients undergoing therapy with TIL and IL-2 has been completed. Several properties of TIL at the time of cell transfer were positively associated with therapeutic response, including shorter doubling times, shorter culture duration, greater autologous tumor lysis, and secretion of GM-CSF by TIL following autologous tumor stimulation. In addition, TIL derived from extranodal metastases were more effective than TIL derived from metastatic lymph nodes. These findings are being used to select candidates most likely to respond to therapy. 2. Generation of a CD8+ T cell clone recognizing the melanoma Ag MART-1. A CD8+ T cell clone was derived from melanoma TIL, which recognized a previously unidentified melanoma-associated Ag. This T cell clone was used by Dr. Y. Kawakami (Surgery Branch, NCI to isolate the gene encoding this Ag, MART-1, and was also used to identify peptides within the MART-1 protein which could be recognized in an HLA-A2 context. Immunization and adoptive cell transfer treatment strategies directed against MART-1 are under development. 3. Identification of a melanoma-associated Ag recognized by CD4+ T cells. We have developed model systems to detect MHC class Il-restricted melanoma associated Ag, using EBV-transformed B cells as Ag presenting cells for these molecules. CD4+ T cells from one melanoma patient recognized a melanoma-associated Ag which was commonly expressed in the patient population, and was also present in cultured normal melanocytes. This Ag was identified as a product of the normal tyrosinase gene, which also encodes class I-restricted epitopes. Identification of commonly expressed tumor associated proteins containing epitopes presented by both MHC class I and class II molecules may provide optimal reagents for cancer immunization strategies.